초록
<P><B>Background</B></P><P>Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx).</P><P><B>Results</B></P><P>We established an <I>Escherichia coli</I> based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, <I>CYP11B1</I> expression was enhanced 3.3-fold to 257 nmol∗L<SUP>−1</SUP> by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the <I>Adx</I> cDNA in order to enhance <I>Adx</I> expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library.</P><P><B>Conclusions</B></P><P>Under optimized conditions a maximum productivity of 0.84 g cortisol∗L<SUP>−1</SUP>∗d<SUP>−1</SUP> was achieved, which clearly shows the potential of the developed system for application in the pharmaceutical industry.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (doi:10.1186/s12934-015-0209-5) contains supplementary material, which is available to authorized users.</P>